Mutation of genes that regulate the cell cycle is a fundamental mechanism underlying tumorigenesis. These mutations involve intrinsic cell cycle components themselves (cyclins, Cdk inhibitors) as well as oncogenes (e.g., c-Myc) and tumor suppressors (e.g. p53, Fbw7) that impact upon the cell cycle machinery. This proposal focuses on the functions and regulation of these cancer-associated cell cycle pathways. The Fbw7 tumor suppressor targets cyclin E, Notch, and c-Jun for degradation after they have been phosphorylated. In preliminary studies, we found that Fbw7 also regulates phosphorylation-dependent c-Myc turnover and that the three Fbw7 isoforms (Fbw7alpha, Fbw7beta, Fbw7gamma) exhibit unique subcellular Iocalizations. In Aim 1 we will test the hypothesis that the Fbw7 isoforms perform distinct biologic functions in these compartments by developing conditional-null mutations of Fbw7alpha and Fbw7gamma in the mouse. We will examine the roles of each isoform in regulating specific Fbw7 substrates, and we will test the hypothesis that Fbw7gamma, regulates c-Myc function in the nucleolus. In Aim 2 we will determine if Fbw7alpha or Fbw7gamma are tumor suppressors. These experiments will define the normal and neoplastic functions of Fbw7gamma and Fbw7alpha. Mutations affecting threonine 58 (T58) are the most common c-Myc mutations in lymphomas and T58 phosphorylation regulates c-Myc stability. We have found that T58 phosphorylation by GSK-3 regulates Fbw7-mediated c-Myc turnover. In Aim 3 we will test the hypothesis that T58 mutations contribute to c-Myc associated neoplasia by preventing the interaction of c-Myc with Fbw7. We will develop knock-in mice in which T58 is mutated that will than be used to study the role of T58 phosphorylation in regulating c-Myc abundance and function, and to determine its role in c-Myc-associated tumorigenesis. Reduced expression of the p27 Cdk inhibitor in human cancers connotes poor prognosis, and p27 is a tumor suppressor in mice. In the last funding period we used insertional mutagenesis in p27-null mice to identify oncogenes that cooperate with p27-1oss, and identified three candidate oncogenes (c-myc, Jdpl, GPC3/XpcI1). The goals of Aim 4 are to utilize murine transplant models to determine the consequences of deregulated Jdp2 and GPC3/Xpcl1 expression in blood cells, to study the cooperativity between Jdp2 or GPC3/Xpcl1 activation and p27-loss, and to understand the mechanisms of this cooperativity.